Ruzzini_2013_Biochemistry_52_7428

Reference

Title : A substrate-assisted mechanism of nucleophile activation in a ser-his-asp containing C-C bond hydrolase - Ruzzini_2013_Biochemistry_52_7428
Author(s) : Ruzzini AC , Bhowmik S , Ghosh S , Yam KC , Bolin JT , Eltis LD
Ref : Biochemistry , 52 :7428 , 2013
Abstract :

The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (betanuc approximately 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

PubMedSearch : Ruzzini_2013_Biochemistry_52_7428
PubMedID: 24067021
Gene_locus related to this paper: sphww-a5j2a5

Related information

Substrate HOPDA-5,8-diF    HODA-6-phenyl
Gene_locus HOPDA-5,8-diF    HODA-6-phenyl    sphww-a5j2a5
Structure HOPDA-5,8-diF    HODA-6-phenyl    sphww-a5j2a5    4LXI    4LYE

Citations formats

Ruzzini AC, Bhowmik S, Ghosh S, Yam KC, Bolin JT, Eltis LD (2013)
A substrate-assisted mechanism of nucleophile activation in a ser-his-asp containing C-C bond hydrolase
Biochemistry 52 :7428

Ruzzini AC, Bhowmik S, Ghosh S, Yam KC, Bolin JT, Eltis LD (2013)
Biochemistry 52 :7428